Stem cell research has become an important field of study for molecular, cellular, and clinical biology as well as pharmaco-toxicology, because stem cells have a strong proliferative and unlimited self-renewal potential and are multipotent. Evidence has suggested that progenitor cells outside the central nervous system, bone marrow cells in particular, may have the ability to generate either neurons or glia in vivo. Toma et al., Nat. Cell Biol. 3:778-783 (2001); Mezey, E. et al., Science 290:1779-1782 (2000); Brazleton, T. R. et al., Science 290:1775-1779 (2000); and Eglitis, M. A. et al., Proc Natl. Acad. Sci. 94:4080-4085 (1997).
Adult bone marrow stromal cells are rare heterogeneous cells including multipotent mesenchymal stromal cells (MSC), adventitial reticular cells, vascular pericytes, and bone-lining cells (Jones, E. & McGonagle, D. Rheumatology (Oxford) 47, 126-31 (2008); Prockop, D. J. Mol. Ther. 17, 939-46 (2009)). These cells are capable of self-renewal, and able to transcribe genes for multiple embryonic germ layers. (Labat, M. L. et al., Biomed. Pharmacother. 54, 146-62 (2000); Woodbury, D. et al., J. Neurosci. Res. 69, 908-17 (2002)). In vivo as well as in vitro studies have confirmed the differentiation of adult bone marrow stem cells into muscle cells, adipocytes, cardiomyocytes, neuroectodermal cells, osteoblasts, chondroblasts, and so on. Recently, it was shown that, under appropriate culture conditions, adult bone marrow stem cells may also differentiate into hepatocyte-like cells, which demonstrates the high potential of adult bone marrow stem cells being used as an unlimited source of hepatocytes for pharmaco-toxicological research and testing. Snykers, S. et al., Methods in Molecular Biology (2006).
However, the identification and isolation of stem cells are challenging, mainly for two reasons. First, stem cells are rare. In bone marrow, for example, where hematopoiesis occurs, there is only one stem cell for every several billion bone marrow cells. Vogel, G. Science, 287, 1418-1419 (2000). Second, it is difficult to identify molecular markers which are unique to stem cells, especially because primitive stem cells may be in a quiescent state and thus may express few molecular markers. Gage, F. H. Science, 287, 1433-1488 (2000).
Some isolation methods of bone marrow stem cells have been reported recently. For example, density-gradient centrifugation is used to isolate murine hematopoietic stem cells on the basis of functional characteristics such as the ability of stem cells to home to bone marrow and aldehyde dehydrogenase (ALDH) activity. An essential component of this method is the separation of whole bone marrow into small-sized cells by counter-flow elutriation instead of the normal elutriation. Juopperi, T. A. et al., Exp Hematol. 2007 February; 35(2):335-41. Recently, a new method for isolation of bone marrow derived liver stem cells (BDLSC) was reported, which involves using a cholestatic serum as the selecting culture system to purify BDLSC directly from bone marrow cells. Cai, Y. F. et al. World J. Gastroenterol 2009; 15(13): 1630-1635. The results suggest that BDLSCs can be purified and passaged. All references cited herein are hereby incorporated by reference in their entirety.
Despite the limited success reported, isolation of bone marrow stem cells remains to be challenging; therefore, development of new methods for isolating bone marrow stem cells is much needed for the fast growing stem cell research and development, and in particular, for the applications of stem cells in cell therapies.
U.S. Pat. No. 6,733,433 discloses a blood separation method particularly for concentrating hematopoietic stem cells. U.S. Pat. No. 5,879,318 similarly discloses methods and closed system for isolating and processing umbilical cord blood. Finally, U.S. Pat. No. 7,279,331 discloses similar methods of isolating cord matrix mesenchymal stem cells from cord fragments. However, none of the three methods allow for the separation of adherent bone marrow or cord blood cells in the same vessel or bag.